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Flow cytometric human leukocyte antigen-B27 typing with stored samples for batch testing
1Department of Clinical Pathology, Kyungpook National University School of Medicine, Daegu, Korea.
Insights
Frozen platelets (FPLT) offer a superior method for flow cytometry HLA-B27 typing using stored blood samples. This approach provides high accuracy and efficiency for diagnosing spondyloarthropathies.
Area of Science:
- Immunology
- Clinical Diagnostics
- Hematology
Background:
- Flow cytometry (FC) is extensively used for diagnosing spondyloarthropathies via HLA-B27 typing.
- Batch testing of stored patient blood samples can improve efficiency and control procurement.
- Investigating optimal storage methods for FC HLA-B27 typing is crucial.
Purpose of the Study:
- To evaluate different patient blood sample storage methods for flow cytometry HLA-B27 typing.
- To determine the most effective method for preserving sample integrity and diagnostic accuracy.
Main Methods:
- Four storage methods were compared: whole blood at room temperature, frozen mononuclear cells, frozen white blood cells (WBCs), and frozen platelets (FPLT).
- Mean fluorescence intensity (MFI) ratios (B27 MFI ratio, B7 MFI ratio, and B7/B27 ratio) were used for HLA-B27 and HLA-B7 assignment.
Main Results:
- Frozen platelets (FPLT) demonstrated superior performance compared to other storage methods.
- FPLT achieved 100% sensitivity and 99.3% specificity for HLA-B27 assignment in DNA-typed samples (N=164) using specific MFI ratio criteria.
Conclusions:
- The FPLT method provides a simple, economical, and accurate approach for FC HLA-B27 typing with stored samples.
- This method has the potential to enhance standard FC typing when combined with DNA-based methods for spondyloarthropathy diagnosis.
Background:
Flow cytometry (FC) HLA-B27 typing is still used extensively for the diagnosis of spondyloarthropathies. If patient blood samples are stored for a prolonged duration, this testing can be performed in a batch manner, and in-house cellular controls could easily be procured. In this study, we investigated various methods of storing patient blood samples.
Methods:
WE COMPARED FOUR STORAGE METHODS: three methods of analyzing lymphocytes (whole blood stored at room temperature, frozen mononuclear cells, and frozen white blood cells [WBCs] after lysing red blood cells [RBCs]), and one method using frozen platelets (FPLT). We used three ratios associated with mean fluorescence intensities (MFI) for HLAB27 assignment: the B27 MFI ratio (sample/control) for HLA-B27 fluorescein-5-isothiocyanate (FITC); the B7 MFI ratio for HLA-B7 phycoerythrin (PE); and the ratio of these two ratios, B7/B27 ratio.
Results:
Comparing the B27 MFI ratios of each storage method for the HLA-B27+ samples and the B7/B27 ratios for the HLA-B7+ samples revealed that FPLT was the best of the four methods. FPLT had a sensitivity of 100% and a specificity of 99.3% for HLA-B27 assignment in DNA-typed samples (N=164) when the two criteria, namely, B27 MFI ratio >4.0 and B7/B27 ratio <1.5, were used.
Conclusions:
The FPLT method was found to offer a simple, economical, and accurate method of FC HLA-B27 typing by using stored patient samples. If stored samples are used, this method has the potential to replace the standard FC typing method when used in combination with a complementary DNA-based method.
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